1. Field of Invention
The present invention relates to novel mutant enzymes which synthesize linear prenyl diphosphates that are precursors of compounds, important to organisms, such as steroids, ubiquinones, dolichols, carotenoids, prenylated proteins, animal hormones, plant hormones, and the like, or a gene thereof etc.
2. Related Art
Of the substances having important functions in the body, many substances are biosynthesized using isoprene (2-methyl-1,3-butadiene) as a building block. These compounds are called isoprenoids, terpenoids, or terpenes, and are classified depending on the number of carbon atoms into hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40), and the like. The actual synthesis starts with the mevalonate pathway through which mevalonic acid-5-diphosphate is synthesized, followed by the synthesis of isopentenyl diphosphate (IPP) which is an active isoprene unit.
The identity of the isoprene unit that was proposed as an speculated precursor was found to be IPP, the so-called active isoprene unit. Dimethylallyl diphosphate (DMAPP), an isomer of IPP, being used as a substrate in the reaction of isopentenyl adenine, which is known as a cytokinin and is one of the plant hormones, is also known to undergo condensation reaction with IPP to synthesize linear active isoprenoids such as geranyl diphosphate (GP), neryl diphosphate, farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP), hexaprenyl diphosphate (HexPP), heptaprenyl diphosphate (HepPP), and the like.
There are Z-type and E-type condensation reactions. GPP is a product of E-type condensation and neryl diphosphate is a product of Z-type condensation. Although, the all-E-type is considered to be the active form in FPP and GGPP, the Z-type condensation reactions lead to the synthesis of various polyprenols found in natural rubber, dolichols, bactoprenols (undecaprenols), and plants. They are believed to undergo the condensation reaction using the phosphate ester bond energy of the pyrophosphate and/or the carbon backbone present in the molecule to produce pyrophosphate and/or phosphate as the byproduct of the reaction.
FPP or GGPP serves as a reaction substrate leading to the synthesis of prenylated proteins (from FPP or GGPP) represented by G-proteins that are important in the mechanism of signal transduction in the cell; cell membrane lipids (from GGPP) of archaea; squalene (from FPP) which is a precursor of steroids; and phytoene (from GGPP) which is a precursor of carotenoid. Prenyl diphosphates from HexPP and HepPP having six and seven isoprene units respectively to prenyl diphosphates having ten isoprene units serve as the precursor of synthesis of ubiquinone and menaquinone (vitamin K2) that work in the electron transport system.
Furthermore, via the biosynthesis of these active-form isoprenoids, the following planty kinds of compounds that are vital to life have been synthesized. Just to mention a few, there are plant hormones of cytokinins and isopetenyl adenosine-modified tRNA that use hemiterpenes as their precursor for synthesis, monoterpene geraniol and the nerol isomers thereof that are the main components of rose oil perfume, and a camphor tree extract camphor which is an insecticide. Sesquiterpens include juvenile hormones of insects, diterpenes include a plant hormone gibberellin, trail pheromones of insects, and retinols and retinals that function as the visual pigment precursors, binding components of the purple membrane proteins of halophilic archaea, and vitamin A.
Furthermore, using squalene, a triterpene, a variety of steroid compounds have been synthesized, including, for example, animal sex hormones, vitamin D, ecdysone which is an mating hormone of insects, a plant hormone brassinolide, and components of plasma membranes. Various carotenoids of tetraterpenes that are precursors of various pigments of organisms and vitamin A are also important compounds derived from active isoprenoids. Compounds such as hlorophyll, pheophytin, tocopherol (vitamin E), and phylloquinone (vitamin K1) are also derived from tetraterpenes.
The active isoprenoid synthases that consecutively condense IPP with such allylic substrates DMAPP, GPP, FPP, GGPP, GFPP, and the like are called prenyl diphosphate synthases, and are also named, based on the maximum chain length of the major reaction products, for example farnesyl diphosphate synthase (FPP synthase), geranylgeranyl diphosphate (GGPP synthase), and the like. There are reports on purification, activity measurement, gene cloning, and its nucleotide sequencing of enzymes such as farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase, heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase, nonaprenyl diphosphate synthase (solanesyl diphosphate synthase), undecaprenyl diphosphate synthase, and the like from bacteria, archaea, fungi, plants, and animals.
These active isoprenoid synthases constituting the basis of synthesis of a great variety of compounds that are important both in the industry and in the field of life sciences have attracted little attention regarding their industrial applications due to their unstable character and low specific activities. However, with the isolation of the genes of FPP synthase and GGPP synthase from thermophilic bacteria and archaea [A. Chen and D. Poulter (1993) J. Biol. Chem. 268: 11002-11007, T. Koyama et al. (1993) J. Biochem. 113: 355-363, S. -i, Ohnuma et al. (1994) J. Biol. Chem. 269: 14792-14797], their availability as enzymes has increased.
The enzymes that synthesize prenyl diphosphates having 20 to 25 carbons are homodimers and are relatively easy to be reacted in vitro, as have been published in many reports. However, the enzymes that synthesize prenyl diphosphates having chain lengths exceeding the above-mentioned length are believed to be heterodimers, or to require additional factors such as a lipid, and the like. Therefore, in order to realize industrial application thereof, it was necessary to find optimal conditions that permit reassembly of two kinds of subunits or additional factors, which was a difficult task.
Therefore, there has been a need for the technology that enables to make the homodimer-type thermostable prenyl diphosphate synthases capable of synthesizing prenyl diphosphates having a longer chain length, by artificially altering the amino acid sequence of the homodimer type prenyl diphosphate synthases that are stable and have high specific activity derived from a thermophilic organism.
As for the prenyl diphosphate synthases derived from thermophilic organisms, there are at present examples of the altered FPP synthase derived from Bacillus stearothermophilus and GGPP synthase derived from Sulfolobus acidocaldarius. The mutant enzyme of FPP synthase of Bacillus stearothermophilus and the gene thereof were selected based on the color change of the organism by lycopene produced by coexistence of crtB (the gene of phytoene synthase) and crtI (the gene of phytoene desaturase, cis:trans isomerase) derived from Erwinia uredovora and the gene of FPP synthase of the mutant B. stearothermophilus in Escherichia coli. GGPP synthase and its mutant and the gene thereof of S acidocaldarius were selected based on the activity of complementing the glycerol metabolic activity of the HexPP synthase-deficient budding yeast of Saccharomyces cereviceae.
The coexistence method of the CrtB and CrtI genes of E. uredovora cannot be used for screening the reaction products longer than GGPP of the mutant enzyme, and the screening method using the complementation activity of the HexPP synthase-deficient budding yeast Saccharomyces cereviceae cannot be used for specific detection of the reaction products longer than HexPP. These genetic screening methods are capable of cloning the genes of the mutant prenyl diphosphate synthases having the synthetic activities of GGPP, GFPP, and HexPP, but cannot systematically control the chain length of the reaction products of prenyl diphosphate synthases with the intention of extending the chain length of the reaction products. A rule for that purpose is not known, either.